The Discovery Of Site Directed Mutagenesis

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

In this experiment, you will model the effects of mutations on the genetic code. Some mutations cause no structural or functional change to proteins while others can have devastating affects on an organism.

Originally in Takahashi and Yamanaka’s experiment, retroviruses were used as a vector to introduce factors that triggers reprogramming, however with evidences from further researches shown that retroviruses may cause insertional mutagenesis, where the host’s DNA is mutated due to the virus integrating into the host’s genome, and potentially cancerous nature of the iPSCs made this technique ill suited to further iPSC developments.(5) Therefore, other methods have been developed and used nowadays to minimise these issues and they include protein transduction, the use of

The EZH2 (enhance of zeste homolog2) is an enzyme that in humans is fixed by EZH2 and its supply the information’s about making of enzyme called a histone methyltransferase In this experiment PCR2 was examined whilst the EZH2 contributes to chemical modification. This resulted in repression. The aim of the study was to re-clone the EZH2 gene from 5’ to 3’ into another plasmid with pBluescript II KS fusing together with EZH2 moving forward. The histidine tags originate in the plasmid. Methods used encountered E.coli transformation, PCR, restriction enzymes, plasmid mini-preps and agarose gel electrophoresis. The end result lead to a 3’ to 5’ of EZH2 inserts. There was a good link which showed that the returning agar plate ensured that the collection of colonies consisted of forward oriented inserts.

The Spot-Overlay Ames Test was used in the lab to find the mutagenesis of Diet Coke and ThermaFlu. Along with these substances three mutant strains of salmonella were also tested. TA 1535, TA 1537, TA 1538 all lacked the ability to grow the amino acid histidine unless reverted back by the potential mutagens. After the first week of testing, results showed that both of the potential mutagens, Diet Coke and ThermaFlu were in fact mutagenic. This was established if colonial growth was at least twice as much as that of the negative control. For the continuation of the experiment, Diet Coke and the TA 1535 strain of S. thyphimurium were chosen to continue experimentation for the second week. Testing commenced once both substances had been mixed

The concept of intentionally altering an organism’s DNA in order to produce genetically modified organisms (GMOs) has been critically analyzed by both science and the mainstream media in the last decade. However, the genetic modification of organisms is not a recent innovation as humans have been modifying organisms for over 30 000 years (Rangel, 2015). Back then, breeding through artificial selection was the most prominent method of genetic engineering. Organisms with the most desirable traits would be bred together in order to create a new generation of superior organisms. Throughout the centuries, the developments in science and technology have resulted in new methods of genetic engineering. Now, genomes can be spliced in order to insert or remove genes. It has essentially come down to a copy and paste process with genes from animals being inserted into plants as the common practice.

Some chemicals can induce the modification of bases: Note: Conversion of C to U will result in a C:G to A:T mutation after replication

For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).

To mutagenize human Kif5A (WT), we performed PCR-mediated site-directed mutagenesis to insert our mutation into the plasmid vector (shown to the right). Next, we performed a restriction enzyme digest with Dpn1, to digest the parental plasmid.  The presence of the PCR product was confirmed by performing gel electrophoresis where the expected band of 6.8 kilobases was obtained. The remaining DNA was used in a bacterial transformation of DH5α to grow colonies. Liquid cultures were grown from overnight colonies and Minipreps were performed to purify the plasmid DNA. These plasmids were sent for sequencing, where the P278L mutation was confirmed.

Throughout the years scientists and researchers have done many studies that pertain to how mutagens cause mutations in genes. They have also studied these mutations to see if they transfer to the next generation or whether the DNA repairs the mutation before it gets passed on. In this lab we will be looking at the mutagen ethyl methanesulfonate (EMS) and how it will affect our organism Caenorhabditis elegans (C. elegans). EMS is known to be mutagenic and carcinogenic because it can “produce significant levels of alkylation at oxygens of guanine and in the DNA phosphate groups. It can also produce GC to AT transition mutations and vice versa. There has also been evidence that EMS is able to break chromosomes” (Sega). We hypothesize that when we expose the C. elegans to the EMS, a mutation will occur on the DNA that will cause a phenotype to occur in the organism that is different from the wild type. Our goal is to be able to locate the mutation on a gene, and match it to other kingdoms to see if they are homologous to other genes. This is beneficial to us as humans

The discovery of the gene transfer mechanisms could be attributed by the work of Lederberg and Tatum back in 1946. Using Escherichia coli(E.coli) as their model, they proposed the genetic materia of E.coli could be exhanged via sexual process. In order to prove their hypothesis, they mutated 2 wild type E.coli strains(K12) using X-ray or ultra-violet radiation to produce Y-10 and Y-24 mutant strains. The former was auxotrophic to threonine, leucine and thiamin whereas the latter failed to produce biotin, phenylalanine and cystine[1]. These mutant could only survive in mininal media plates provided that the aforementioned amino acids are supplied accordingly to each mutant strains.

This was done by using DNA ligation and E.Coli transformation. We looked at agar plates to analyze which one of E.coli cell strains took up the vector alone of the vector containing the gene of interest. Four agar plates were used in this laboratory which were labeled Ligation, pGEM- T Positive Control, Competent Cells Negative Control, and Competent Cells Positive Control. Reagents were used such as DNA Ligase Buffer, SOC Media, Ampicillin, IPTG, and X-Gal. Ligation was performed using 2X ligation buffer, DNA Mix, T4 DNA Ligase and sterile dH2O. Then the transformation of E. coli occurred in which our 4 LB agar plates were prepared with the corresponding amount of IPTG, X-Gal and ampicillin gene. The next step was pipetting the content into the corresponding plates and SOC media was added.The transformation tubes consent were placed on the agar plates using a spreader. The plates were incubated at 37 Celsius and then will be stored at 4 degrees Celsius.The ligation plate was prepared by adding 100 µL of IPTG, 50 µL of X-Gal and Ampicillin. In the ligation plate, the expected color to be see was blue. However, in our ligation plate, we were seeing both blue and white colonies. Blue cells indicate that the cells take up the vector alone in the presence of IPTG and X-Gal due to Beta- Galactosidase expression. IPTG or

Genetic engineering is the deliberate modification of the characteristics of an organism by manipulating its deoxyribonucleic acid (DNA). Bacteria possess plasmids which can be used as vectors for bacterial transformation. The plasmids can be altered to include donor DNA thus altering the physiological or physical characteristics of the bacterium. Escherichia coli have been found to be the most effective bacterium in genetic transformation and heterologous expression of human genes or proteins. It was hypothesised that Escherichia coli HB101 K12 could be transformed with a pGLO plasmid to express the genes encoded for green fluorescent protein (GFP), the bla gene for ampicillin resistance and the araC gene a regulatory protein in facilitating transcription and therefore the transformation efficiency for this bacteria could be calculated. Bacillus megaterium, E.coli B strain, Erwinia carotovora, Pseudomonas fluorescens, Staphylococcus epidermidis and Streptococcus lactis will also be subjected to transformation with the pGLO plasmid to collect transformation efficiency data for comparison against Escherichia coli HB101 K12. Escherichia coli HB101 K12 were found to have been successfully transformed with a transformation efficiency falling within the transformation protocol of 8.0 x 102 and 7.0 x 103. The incidence of Escherichia coli HB101 K12 on the LB/amp and LB/amp/ara pGLO+ plates indicated that the bacterial colonies present showed resistance to the ampicillin

With only a 20-year history, genetic engineering is only a young science with much uncertainly relativity compared to other fields. Much of the elements bioengineering studies and experiments, the DNA genome and sequence, are still unknown to the scientific community. Changes in genes greatly transform the condition, structure, and essence of an organism, giving us an entirely different and unnatural creation. Even when the physical effects are barely noticeable or deemed safe and ethical, these modified creations’ effects on the environment are impossible to predict and will only emerge when the damage done becomes evident to the human population. Though a large unknown looms over modifying genes, bioengineering industries insist upon dismissing the scare over genetic foods with the vast potential “miracle” foods can benefit upon human society.

Direct manipulation of DNA focuses on selective breeding, altering organisms to achieve higher quality products and more of them. These improved